Isoprenoid pathway inhibitors for stimulating bone growth

ABSTRACT

Compounds of the formula  
                 
 
     wherein X in each of formulas (1) and (2) represents a substituted or unsubstituted alkylene, alkenylene, or alkynylene linker of 2-6C;  
     Y is of the formula  
                 
 
     or a stereoisomer thereof,  
     wherein R′ is substituted or unsubstituted alkyl;  
     each R 2  is independently H, hydroxy, alkoxy (1-6C) or lower alkyl (1-4C);  
     R 3  is H, hydroxy, or alkoxy (1-6C); or  
     Y is of the formula  
                 
 
     wherein each n is 1,  
     Z is N,  
     K comprises a substituted or unsubstituted aromatic carbocyclic or heterocyclic ring system which may optionally be spaced from the linkage position shown in formula (7) by a linker of 1-2C, or in formula (7), Z may be spaced from the carbon bonded to X by═CR 6 — wherein R 6  is H or linear, branded or cyclic alkyl (1-6C),  
     R 5  is H or linear, branched or cyclic alkyl, and  
     R′ represents a cation, H or a substituted or unsubstituted alkyl group of 1-6C,  
     promote bone formation and are thus useful in treating osteoporosis, bone fracture or deficiency, primary or secondary hyperparathyroidism, periodontal disease or defect, metastatic bone disease, osteolytic bone disease, post-plastic surgery, post-prosthetic joint surgery, and post-dental implantation.  
     Also disclosed is a method to identify additional compounds which are inhibitors of enzymes in the isoprenoid scheme especially of HMG-CoA reductase which results in prenylation of proteins and in the synthesis of steroids or of inhibitors of their production which are useful in treating bone disorders.

[0001] This application claims priority under 35 U.S.C. §119 fromprovisional application Ser. No. 60/032,893 filed Dec. 13, 1996. It isalso a continuation-in-part of U.S. Ser. No. 09/096,631 filed Jun. 12,1998 which is a continuation-in-part of U.S. Ser. No. 08/989,862 filedDec. 12, 1997 claiming benefit under 35 U.S.C. §120. The entire contentsof these documents are incorporated herein by reference.

TECHNICAL FIELD

[0002] The invention relates to screening methods for compositions andto methods to use these compositions in treating bone disorders invertebrates including fractures and cartilage disorders. Morespecifically, the invention concerns a method to identify agents thatwill be useful in treating bone disorders by assessing their ability toinhibit enzymes in the isoprenoid synthesis pathway and in particular,to inhibit HMG-CoA reductase, and to methods and compositions useful intreating bone disorders which contain the identified active ingredientsas essential components.

BACKGROUND ART

[0003] Bone is subject to constant breakdown and resynthesis in acomplex process mediated by osteoblasts, which produce new bone, andosteoclasts, which destroy bone. The activities of these cells areregulated by a large number of cytokines and growth factors, many ofwhich have now been identified and cloned.

[0004] There is a plethora of conditions which are characterized by theneed to enhance bone formation. Perhaps the most obvious is the case ofbone fractures, where it would be desirable to stimulate bone growth andto hasten and complete bone repair. Agents that enhance bone formationwould also be useful in facial reconstruction procedures. Other bonedeficit conditions include bone segmental defects, periodontal disease,metastatic bone disease, osteolytic bone disease and conditions whereconnective tissue repair would be beneficial, such as healing orregeneration of cartilage defects or injury. Also of great significanceis the chronic condition of osteoporosis, including age-relatedosteoporosis and osteoporosis associated with post-menopausal hormonestatus. Other conditions characterized by the need for bone growthinclude primary and secondary hyperparathyroidism, disuse osteoporosis,diabetes-related osteoporosis, and glucocorticoid-related osteoporosis.

[0005] One group of compounds suggested for enhancing bone formationcomprises bone morphogenic proteins (BMPs). The BMPs are novel factorsin the extended transforming growth factor β superfamily. RecombinantBMP-2 and BMP-4 can induce new bone formation when they are injectedlocally into the subcutaneous tissues of rats (Wozney J. Molec ReprodDev (1992) 32:160-67). These factors are expressed by normal osteoblastsas they differentiate, and have been shown to stimulate osteoblastdifferentiation and bone nodule formation in vitro as well as boneformation in vivo (Harris S., et al., J. Bone Miner Res (1994)9:855-63). This latter property suggests potential usefulness astherapeutic agents in diseases which result in bone loss.

[0006] The cells which are responsible for forming bone are osteoblasts.As osteoblasts differentiate from precursors to mature bone-formingcells, they express and secrete a number of enzymes and structuralproteins of the bone matrix, including Type-1 collagen, osteocalcin,osteopontin and alkaline phosphatase (Stein G., et al., Curr Opin CellBiol (1990) 2:1018-27; Harris S., et al., (1994), supra). They alsosynthesize a number of growth regulatory peptides which are stored inthe bone matrix, and are presumably responsible for normal boneformation. These growth regulatory peptides include the BMPs (Harris S.,et al. (1994), supra). In studies of primary cultures of fetal ratcalvarial osteoblasts, BMPs 1, 2, 3, 4, and 6 are expressed by culturedcells prior to the formation of mineralized bone nodules (Harris S., etal. (1994), supra). Like alkaline phosphatase, osteocalcin andosteopontin, the BMPs are expressed by cultured osteoblasts as theyproliferate and differentiate.

[0007] Although the BMPs are potent stimulators of bone formation invitro and in vivo, there are disadvantages to their use as therapeuticagents to enhance bone healing. Receptors for the bone morphogeneticproteins have been identified in many tissues, and the BMPs themselvesare expressed in a large variety of tissues in specific temporal andspatial patterns. This suggests that BMPs may have effects on manytissues in addition to bone, potentially limiting their usefulness astherapeutic agents when administered systemically. Moreover, since theyare peptides, they would have to be administered by injection. Thesedisadvantages impose severe limitations to the development of BMPs astherapeutic agents.

[0008] Small molecules that are useful in treating bone disorders invertebrates are of the general formula Ar¹-L-Ar² wherein Ar¹ and Ar² arearomatic moieties and L is a linker that separates them by a specifieddistance. These are disclosed in PCT application WO98/17267 publishedApr. 30, 1998. These compounds were assessed for usefulness in treatingbone disorders by their ability to enhance the production of a reporterprotein when the nucleotide sequence encoding the reporter protein isoperably linked to the promoter for BMP-2. Similar compounds aredisclosed for this purpose in earlier filed PCT applications WO97/15308published May 1, 1997 and WO97/48694 published Dec. 24, 1997. Thepresent application concerns another class of compounds that areinhibitors of β-hydroxy-β-methyl glutaric acid CoA (HMG-CoA) reductasethat are also successful in this assay. The compounds described in thepresent application are generically known as “statins.”

[0009] Statins are HMG-CoA reductase inhibitors (Bilheimer, D. W., DrugInvestigation (1900) 2 (Suppl. 2) 58-67). HMG-CoA reductase is theprincipal rate limiting enzyme involved in cellular cholesterolbiosynthesis. The pathway is also responsible for the production ofdolichol, ubiquinones, isopentenyl adenine and farnesol. HMG-CoAreductase converts 3-hydroxy-3-methyl-glutaryl CoA (HMG-CoA) tomevalonate. Addition of mevalonate at concentrations between 25-800 μMinhibits the activity of mevastatin (100, 25, or 6.25 μM) in the ABAassay described in Example 1 herein. Mevalonic acid has no effect onprimary screen activities of bone growth-active compounds outside of thestatin family (compounds 59-0008 (see Example 1)). These data indicatethat the effect of mevastatin in the ABA assay is mediated by its effecton HMG-CoA reductase. Knowledge of inhibitors of the cholesterolbiosynthetic pathway (including SAR or pharmacophore analyses) may beuseful in determining appropriate modifications or analogs of thestatins that maintain bone growth activity.

[0010] U.S. Pat. No. 5,280,040 discloses compounds described as usefulin the treatment of osteoporosis. These compounds putatively achievethis result by preventing bone resorption. Related to these compoundsare the bisphosphonates-the methylene bisphosphonic acids. Thesecompounds are comprised of two phosphonic acid residues coupled througha methylene linkage. Typical representatives include the clodronateswhich are simple compounds wherein the phosphonic acid residues arecoupled through dichloromethylene. Other representative bisphosphonatesinclude ibandronates, the risedronates, alandronates and pamidronates.These compounds have been shown to inhibit the resorption of bone,presumably by effecting apoptosis of osteoclasts. Luckman, S. P., etal., J Bone Min Res (1998) 13:581-589.

[0011] Wang, G.-J., et al., J Formos Med Assoc (1995) 94:589-592 reportthat certain lipid clearing agents, exemplified by lovastatin andbezafibrate, were able to inhibit the bone resorption resulting fromsteroid administration in rabbits. There was no effect on bone formationby these two compounds in the absence of steroid treatment. Themechanism of the inhibition in bone resorption observed in the presenceof steroids (and the mechanism of the effect of steroid on bone per se)is said to be unknown. The authors state that steroid-induced bone lossis associated with a decrease in bone formation attributed to aninhibitory effect of corticosteroid on osteoblast activity and anincrease in bone absorption due to direct osteoclast stimulation and toan indirect inhibition of intestinal calcium absorption with a secondaryincrease in parathyroid hormone production. Other mechanisms mentionedinclude those attributable to lipid abnormalities and hyperlipidemiawhich lead to circulatory impairment, obstruction of subchondralvessels, osteocyte necrosis and osteoporosis. In light of the knownactivities of lovastatin and bezafibrate, the authors attribute theeffect on bone loss to their ability to lower lipid levels and overcomethe impairment to circulation within the femoral head. There is nosuggestion in Wang, et al., that lovastatin directly enhances boneformation.

[0012] An abstract entitled “Lovastatin Prevents Steroid-InducedAdipogenesis and Osteoporosis” by Cui, Q., et al., appeared in theReports of the ASBMR 18th Annual Meeting (September 1996) J. BoneMineral Res. (1996) 11(S1):S510. The abstract reports that lovastatindiminished triglyceride vesicles that accumulated when osteoprogenitorcells cloned from bone marrow stroma of chickens were treated in culturewith dexamethasone. Lovastatin was reported to diminish the expressionof certain mRNAs and to allow the cells to maintain the osteogenicphenotype after dexamethasone treatment. Further, chickens that hadundergone bone loss in the femoral head as a result of dexamethasonetreatment were improved by treatment with lovastatin. Again, there is nosuggestion that lovastatin directly enhances bone formation in theabsence of steroid treatment.

[0013] In any event, these data are contrary to reports thatdexamethasone and other inducers, such as BMPs, induce osteoblasticdifferentiation and stimulate osteocalcin mRNA (Bellows, C. G., et al.,Develop Biol (1990) 140:132-38; Rickard, D. J., et al., Develop Biol(1994) 161:218-28). In addition, Ducy, P., et al., Nature (1996)382:448-52 have recently reported that osteocalcin deficient miceexhibit a phenotype marked by increased bone formation and bones ofimproved functional quality, without impairment of bone resorption.Ducy, et al., state that their data suggest that osteocalcin antagonistsmay be of therapeutic use in conjunction with estrogen replacementtherapy (for prevention or treatment of osteoporosis).

[0014] The present invention discloses not only the class of compoundsgenerally called the “statins” for use in stimulating bone formation,but also provides a method to identify compounds useful in this regardby assessing their ability to inhibit enzymes in the pathway ofisoprenoid synthesis. These enzymes include HMG-CoA reductase (inhibitedby the statins), and also the enzymes responsible for production of thegeranyl and famesyl intermediates on the pathway to the synthesis ofsqualene and ultimately the steroids and the enzymes which catalyze theaddition of farnesyl units or geranyl-geranyl units to proteins.

DISCLOSURE OF THE INVENTION

[0015] In one aspect, the invention provides statin-type compounds thatcan be administered as ordinary pharmaceuticals and have the metaboliceffect of directly enhancing bone growth. The statins can be confirmedin this property using an assay for their ability to activate controlelements associated with endogenous factors that stimulate bone growth.Thus, the invention is directed to methods and compositions forstimulating the growth of skeletal (bone) tissue, which methods andcompositions use, as at least one of the active ingredients, compoundswhich are characterized as “statins” by virtue of their ability toinhibit HMG-CoA reductase. Typical statins are of the formula:

[0016] wherein X in each of formulas (1) and (2) represents asubstituted or unsubstituted alkylene, alkenylene, or alkynylene linkerof 2-6C;

[0017] Y represents one or more carbocyclic or heterocyclic rings; whentwo or more rings are present in Y, they may optionally be fused; and

[0018] R′ represents a cation, H or a substituted or unsubstituted alkylgroup of 1-6C.

[0019] Thus, the invention is directed to methods to treat bonedisorders by directly stimulating bone formation using the statinsdescribed and to pharmaceutical compositions for this use.

[0020] In still another aspect, the invention is directed to methods toidentify additional compounds which are useful in methods to treat bonedisorders by assessing their ability to inhibit enzymes significant inthe isoprenoid synthesis pathway, especially HMG-CoA reductase, but alsoincluding subsequent steps resulting in the synthesis of steroids andsubsequent steps resulting in the prenylation of proteins. Compoundsidentified in this manner can be confirmed as useful in treating bonedisorders in any of a number of assays described hereinbelow. Theinvention is also directed to methods to treat bone disorders usingcompounds that are inhibitors of the isoprenoid and isoprenoid-relatedpathways.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021]FIGS. 1A and 1B show the structures and activity of severalcompounds of the invention in the ABA screening assay of Example 1.

[0022]FIG. 2 shows an outline of the synthesis pathway for isoprenoidsand the pathways of their subsequent conversion to squalene and steroidsand in prenylating target proteins.

MODES OF CARRYING OUT THE INVENTION

[0023] The ultimate goal of the methods and compositions of theinvention is to treat or ameliorate bone disorders in vertebratesubjects, particularly mammals, and more particularly humans.

[0024] As used herein, “treat” or “treatment” include a postponement ofdevelopment of bone deficit symptoms and/or a reduction in the severityof such symptoms that will or are expected to develop. The terms furtherinclude ameliorating existing bone or cartilage deficit symptoms,preventing additional symptoms, ameliorating or preventing theunderlying metabolic causes of symptoms, preventing or reversing boneresorption and/or encouraging bone growth. Thus, the terms denote that abeneficial result has been conferred on a vertebrate subject with acartilage, bone or skeletal deficit, or with the potential to developsuch deficit.

[0025] By “bone deficit” is meant an imbalance in the ratio of boneformation to bone resorption, such that, if unmodified, the subject willexhibit less bone than desirable, or the subject's bones will be lessintact and coherent than desired. Bone deficit may also result fromfracture, from surgical intervention or from dental or periodontaldisease. By “cartilage defect” is meant damaged cartilage, lesscartilage than desired, or cartilage that is less intact and coherentthan desired. “Bone disorders” includes both bone deficits and cartilagedefects.

[0026] Representative uses of the compounds of the present inventioninclude: repair of bone defects and deficiencies, such as thoseoccurring in closed, open and non-union fractures; prophylactic use inclosed and open fracture reduction; promotion of bone healing in plasticsurgery; stimulation of bone ingrowth into non-cemented prostheticjoints and dental implants; elevation of peak bone mass inpre-menopausal women; treatment of growth deficiencies; treatment ofperiodontal disease and defects, and other tooth repair processes;increase in bone formation during distraction osteogenesis; andtreatment of other skeletal disorders, such as age-related osteoporosis,post-menopausal osteoporosis, glucocorticoid-induced osteoporosis ordisuse osteoporosis and arthritis, or any condition that benefits fromstimulation of bone formation. The compounds of the present inventioncan also be useful in repair of congenital, trauma-induced or surgicalresection of bone (for instance, for cancer treatment), and in cosmeticsurgery. Further, the compounds of the present invention can be used forlimiting or treating cartilage defects or disorders, and may be usefulin wound healing or tissue repair.

[0027] Bone or cartilage deficit or defect can be treated in vertebratesubjects by administering compounds of the invention which exhibitcertain structural and functional characteristics. The compositions ofthe invention may be administered systemically or locally. For systemicuse, the compounds herein are formulated for parenteral (e.g.,intravenous, subcutaneous, intramuscular, intraperitoneal, intranasal ortransdermal) or enteral (e.g., oral or rectal) delivery according toconventional methods. Intravenous administration can be by a series ofinjections or by continuous infusion over an extended period.Administration by injection or other routes of discretely spacedadministration can be performed at intervals ranging from weekly to onceto three times daily. Alternatively, the compounds disclosed herein maybe administered in a cyclical manner (administration of disclosedcompound; followed by no administration; followed by administration ofdisclosed compound, and the like). Treatment will continue until thedesired outcome is achieved. In general, pharmaceutical formulationswill include a compound of the present invention in combination with apharmaceutically acceptable vehicle, such as saline, buffered saline, 5%dextrose in water, borate-buffered saline containing trace metals or thelike. Formulations may further include one or more excipients,preservatives, solubilizers, buffering agents, albumin to preventprotein loss on vial surfaces, lubricants, fillers, stabilizers, etc.Methods of formulation are well known in the art and are disclosed, forexample, in Remington's Pharmaceutical Sciences, latest edition, MackPublishing Co., Easton Pa., which is incorporated herein by reference.Pharmaceutical compositions for use within the present invention can bein the form of sterile, non-pyrogenic liquid solutions or suspensions,coated capsules, suppositories, lyophilized powders, transdermal patchesor other forms known in the art. Local administration may be byinjection at the site of injury or defect, or by insertion or attachmentof a solid carrier at the site, or by direct, topical application of aviscous liquid, or the like. For local administration, the deliveryvehicle preferably provides a matrix for the growing bone or cartilage,and more preferably is a vehicle that can be absorbed by the subjectwithout adverse effects.

[0028] Delivery of compounds herein to wound sites may be enhanced bythe use of controlled-release compositions, such as those described inPCT application WO 93/20859, which is incorporated herein by reference.Films of this type are particularly useful as coatings for prostheticdevices and surgical implants. The films may, for example, be wrappedaround the outer surfaces of surgical screws, rods, pins, plates and thelike. Implantable devices of this type are routinely used in orthopedicsurgery. The films can also be used to coat bone filling materials, suchas hydroxyapatite blocks, demineralized bone matrix plugs, collagenmatrices and the like. In general, a film or device as described hereinis applied to the bone at the fracture site. Application is generally byimplantation into the bone or attachment to the surface using standardsurgical procedures.

[0029] In addition to the copolymers and carriers noted above, thebiodegradable films and matrices may include other active or inertcomponents. Of particular interest are those agents that promote tissuegrowth or infiltration, such as growth factors. Exemplary growth factorsfor this purpose include epidermal growth factor (EGF), fibroblastgrowth factor (FGF), platelet-derived growth factor (PDGF), transforminggrowth factors (TGFs), parathyroid hormone (PTH), leukemia inhibitoryfactor (LIF), insulin-like growth factors (IGFs) and the like. Agentsthat promote bone growth, such as bone morphogenetic proteins (U.S. Pat.No. 4,761,471; PCT Publication WO 90/11366), osteogenin (Sampath, etal., Proc. Natl. Acad. Sci. USA (1987) 84:7109-13) and NaF (Tencer, etal., J. Biomed. Mat. Res. (1989) 23: 571-89) are also contemplated.Biodegradable films or matrices include calcium sulfate, tricalciumphosphate, hydroxyapatite, polylactic acid, polyanhydrides, bone ordermal collagen, pure proteins, extracellular matrix components and thelike and combinations thereof. Such biodegradable materials may be usedin combination with non-biodegradable materials, to provide desiredmechanical, cosmetic or tissue or matrix interface properties.

[0030] Alternative methods for delivery of compounds of the presentinvention include use of ALZET osmotic minipumps (Alza Corp., Palo Alto,Calif.); sustained release matrix materials such as those disclosed inWang, et al. (PCT Publication WO 90/11366); electrically charged dextranbeads, as disclosed in Bao, et al., (PCT Publication WO 92/03125);collagen-based delivery systems, for example, as disclosed in Ksander,et al., Ann. Surg. (1990) 211(3):288-94; methylcellulose gel systems, asdisclosed in Beck, et al., J. Bone Min. Res. (1991) 6(11):1257-65;alginate-based systems, as disclosed in Edelman, et al., Biomaterials(1991) 12:619-26 and the like. Other methods well known in the art forsustained local delivery in bone include porous coated metal prosthesesthat can be impregnated and solid plastic rods with therapeuticcompositions incorporated within them.

[0031] The compounds of the present invention may also be used inconjunction with agents that inhibit bone resorption. Antiresorptiveagents, such as estrogen, bisphosphonates and calcitonin, are preferredfor this purpose. More specifically, the compounds disclosed herein maybe administered for a period of time (for instance, months to years)sufficient to obtain correction of a bone deficit condition. Once thebone deficit condition has been corrected, the vertebrate can beadministered an anti-resorptive compound to maintain the corrected bonecondition. Alternatively, the compounds disclosed herein may beadministered with an anti-resorptive compound in a cyclical manner(administration of disclosed compound, followed by anti-resorptive,followed by disclosed compound, and the like).

[0032] In additional formulations, conventional preparations such asthose described below may be used.

[0033] Aqueous suspensions may contain the active ingredient inadmixture with pharmacologically acceptable excipients, comprisingsuspending agents, such as methyl cellulose; and wetting agents, such aslecithin, lysolecithin or long-chain fatty alcohols. The said aqueoussuspensions may also contain preservatives, coloring agents, flavoringagents, sweetening agents and the like in accordance with industrystandards.

[0034] Preparations for topical and local application comprise aerosolsprays, lotions, gels and ointments in pharmaceutically appropriatevehicles which may comprise lower aliphatic alcohols, polyglycols suchas glycerol, polyethylene glycol, esters of fatty acids, oils and fats,and silicones. The preparations may further comprise antioxidants, suchas ascorbic acid or tocopherol, and preservatives, such asp-hydroxybenzoic acid esters.

[0035] Parenteral preparations comprise particularly sterile orsterilized products. Injectable compositions may be provided containingthe active compound and any of the well known injectable carriers. Thesemay contain salts for regulating the osmotic pressure.

[0036] If desired, the osteogenic agents can be incorporated intoliposomes by any of the reported methods of preparing liposomes for usein treating various pathogenic conditions. The present compositions mayutilize the compounds noted above incorporated in liposomes in order todirect these compounds to macrophages, monocytes, as well as other cellsand tissues and organs which take up the liposomal composition. Theliposome-incorporated compounds of the invention can be utilized byparenteral administration, to allow for the efficacious use of lowerdoses of the compounds. Ligands may also be incorporated to furtherfocus the specificity of the liposomes.

[0037] Suitable conventional methods of liposome preparation include,but are not limited to, those disclosed by Bangham, A. D., et al., J MolBiol (1965) 23:238-252, Olson, F., et al., Biochim Biophys Acta (1979)557:9-23, Szoka, F., et al., Proc Natl Acad Sci USA (1978) 75:4194-4198,Kim, S., et al., Biochim Biophys Acta (1983) 728:339:348, and Mayer, etal., Biochim Biophys Acta (1986) 858:161-168.

[0038] The liposomes may be made from the present compounds incombination with any of the conventional synthetic or naturalphospholipid liposome materials including phospholipids from naturalsources such as egg, plant or animal sources such asphosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol,sphingomyelin, phosphatidylserine, or phosphatidylinositol and the like.Synthetic phospholipids that may also be used, include, but are notlimited to: dimyristoylphosphatidylcholine, dioleoylphosphatidylcholine,dipalmitoylphosphatidylcholine and distearoylphosphatidycholine, and thecorresponding synthetic phosphatidylethanolamines andphosphatidylglycerols. Cholesterol or other sterols, cholesterolhemisuccinate, glycolipids, cerebrosides, fatty acids, gangliosides,sphingolipids, 1,2-bis(oleoyloxy)-3-(trimethyl ammonio) propane (DOTAP),N-[1-(2,3-dioleoyl) propyl-N,N,N-trimethylammonium chloride (DOTMA), andother cationic lipids may be incorporated into the liposomes, as isknown to those skilled in the art. The relative amounts of phospholipidand additives used in the liposomes may be varied if desired. Thepreferred ranges are from about 60 to 90 mole percent of thephospholipid; cholesterol, cholesterol hemisuccinate, fatty acids orcationic lipids may be used in amounts ranging from 0 to 50 molepercent. The amounts of the present compounds incorporated into thelipid layer of liposomes can be varied with the concentration of thelipids ranging from about 0.01 to about 50 mole percent.

[0039] The liposomes with the above formulations may be made still morespecific for their intended targets with the incorporation of monoclonalantibodies or other ligands specific for a target. For example,monoclonal antibodies to the BMP receptor may be incorporated into theliposome by linkage to phosphatidylethanolamine (PE) incorporated intothe liposome by the method of Leserman, L., et al., Nature (1980)288:602-604.

[0040] Veterinary uses of the disclosed compounds are also contemplated.Such uses would include treatment of bone or cartilage deficits ordefects, i.e., bone disorders, in domestic animals, livestock andthoroughbred horses.

[0041] The compounds of the present invention may be used to stimulategrowth of bone-forming cells or their precursors, or to inducedifferentiation of bone-forming cell precursors, either in vitro or exvivo. The compounds described herein may also modify a target tissue ororgan environment, so as to attract bone-forming cells to an environmentin need of such cells. As used herein, the term “precursor cell” refersto a cell that is committed to a differentiation pathway, but thatgenerally does not express markers or function as a mature, fullydifferentiated cell. As used herein, the term “mesenchymal cells” or“mesenchymal stem cells” refers to pluripotent progenitor cells that arecapable of dividing many times, and whose progeny will give rise toskeletal tissues, including cartilage, bone, tendon, ligament, marrowstroma and connective tissue (see A. Caplan, J. Orthop. Res. (1991)9:641-50). As used herein, the term “osteogenic cells” includesosteoblasts and osteoblast precursor cells. More particularly, thedisclosed compounds are useful for stimulating a cell populationcontaining marrow mesenchymal cells, thereby increasing the number ofosteogenic cells in that cell population. In a preferred method,hematopoietic cells are removed from the cell population, either beforeor after stimulation with the disclosed compounds. Through practice ofsuch methods, osteogenic cells may be expanded. The expanded osteogeniccells can be infused (or reinfused) into a vertebrate subject in needthereof. For instance, a subject's own mesenchymal stem cells can beexposed to compounds of the present invention ex vivo, and the resultantosteogenic cells could be infused or directed to a desired site withinthe subject, where further proliferation and/or differentiation of theosteogenic cells can occur without immunorejection. Alternatively, thecell population exposed to the disclosed compounds may be immortalizedhuman fetal osteoblastic or osteogenic cells. If such cells are infusedor implanted in a vertebrate subject, it may be advantageous to“immunoprotect” these non-self cells, or to immunosuppress (preferablylocally) the recipient to enhance transplantation and bone or cartilagerepair.

[0042] Within the present invention, an “effective amount” of acomposition is that amount which produces a statistically significanteffect. For example, an “effective amount” for therapeutic uses is theamount of the composition comprising an active compound herein requiredto provide a clinically significant increase in healing rates infracture repair; reversal of bone loss in osteoporosis; reversal ofcartilage defects or disorders; prevention or delay of onset ofosteoporosis; stimulation and/or augmentation of bone formation infracture non-unions and distraction osteogenesis; increase and/oracceleration of bone growth into prosthetic devices; and repair ofdental defects. Such effective amounts will be determined using routineoptimization techniques and are dependent on the particular condition tobe treated, the condition of the patient, the route of administration,the formulation, and the judgment of the practitioner and other factorsevident to those skilled in the art. The dosage required for thecompounds of the invention (for example, in osteoporosis where anincrease in bone formation is desired) is manifested as a statisticallysignificant difference in bone mass between treatment and controlgroups. This difference in bone mass may be seen, for example, as a5-20% or more increase in bone mass in the treatment group. Othermeasurements of clinically significant increases in healing may include,for example, tests for breaking strength and tension, breaking strengthand torsion, 4-point bending, increased connectivity in bone biopsiesand other biomechanical tests well known to those skilled in the art.General guidance for treatment regimens is obtained from experimentscarried out in animal models of the disease of interest.

[0043] The dosage of the compounds of the invention will vary accordingto the extent and severity of the need for treatment, the activity ofthe administered compound, the general health of the subject, and otherconsiderations well known to the skilled artisan. Generally, they can beadministered to a typical human on a daily basis as an oral dose ofabout 0.1 mg/kg-1000 mg/kg, and more preferably from about 1 mg/kg toabout 200 mg/kg. The parenteral dose will appropriately be 20-100% ofthe oral dose. While oral administration may be preferable in mostinstances (for reasons of ease, patient acceptability, and the like),alternative methods of administration may be appropriate for selectedcompounds and selected defects or diseases. In comparative assays,positive control compounds or other bone-active test compounds may beadministered subcutaneously, while statin-type test compounds areadministered orally.

[0044] Identification of Statins and Other Isoprenoid Pathway Inhibitors

[0045] The compounds of the invention which are useful in treating bonedisorders by enhancing bone growth are generally classified as“statins.” Statins are known to inhibit the enzyme HMG-CoA reductase.The structure of a number of statins, currently used as drugs to inhibitcholesterol formation, are shown in FIG. 1. Members of this group ofcompounds have found considerable practical application and are marketedas cholesterol-lowering drugs. These include cerivastatin, marketedunder the name Baycol® by Bayer (See U.S. Pat. Nos. 5,006,530 and5,177,080), lovastatin, marketed under the name Mevacor® by Merck (SeeU.S. Pat. No. 4,963,538), and simvastatin, marketed under the name ofZocar®, pravastatin, marketed under the brand name Pravachol®,atorvastatin, marketed under the name Lipotor® by Warner-Lambert (SeeU.S. Pat. No. 5,273,995), and fluvastatin, marketed under the nameLescolo (See U.S. Pat. No. 4,739,073). Another known statin is NK-104developed by NEGMA. (See Akiba, T et al., J Toxicol Sci (1998)23V:713-720.) All the above-cited documents are incorporated herein byreference. In general, additional compounds of similar structure whichare successful in inhibiting HMG-CoA reductase can be identified bystandard assays well known in the art, and as described below.

[0046]FIG. 2 is a diagram of the synthetic pathway which includesisoprenoid intermediates and ultimately results in the formation ofsteroids or the prenylation of proteins. As used herein, “isoprenoidpathway” refers to the conversions summarized in this figure, and“enzymes of the isoprenoid pathway” refers to any enzyme which catalyzesthese conversions. More detailed descriptions of the individualconversions in the general outline shown in FIG. 2 will be found instandard texts on metabolism and biochemistry. The outline in FIG. 2 isintended as an overview only, and does not depict each and everyconversion step. These are as have been elucidated over the past 40 orso years in the study of acetate metabolism. Compounds which inhibitthese pathways, represented by the diagram in FIG. 2, are also useful instimulating bone growth or inhibiting bone resorption or both. Compoundsthat inhibit the various steps in this pathway can easily be identifiedby assessing their ability to inhibit the particular enzymes thatcatalyze the relevant steps. Thus, the invention is also directed to amethod to identify compounds useful in the treatment of bone disorderswhich method comprises determining the ability of a candidate compoundto inhibit an enzymic conversion in this pathway. This assay can beconducted by contacting said compound with an assay mixture for theactivity of an enzyme in the isoprenoid pathway; determining theactivity of the enzyme in the presence as compared to the absence ofsaid compound; wherein a decrease of activity of said enzyme in thepresence as opposed to the absence of said compound indicates that thecompound will be useful in treating bone disorders in vertebrates.

[0047] Since the enzymes involved in this pathway are known and oftencommercially available, simple in vitro assays for this inhibitionactivity are well within ordinary skill.

[0048] In one aspect, the inhibition of the pathway diagrammed in FIG. 2may result in the stimulation of bone formation by preventingprenylation of proteins which are inhibitory to the synthesis of bonewhen in their prenylated form. For example, the function of the rasprotein is dependent on prenylation, and inhibition of the functions ofras protein in bone cells leads to increased bone formation as set forthin Example 7 below.

[0049] Compounds that are identified by their ability to inhibit thevarious enzymes in the isoprenoid synthesis pathway can be confirmed tostimulate bone formation by the use of suitable more direct assays asset forth hereinabove.

[0050] In addition, compounds useful in treating bone disorders can beidentified by their ability to diminish the production of these enzymes.This can be assessed by testing the effect of a candidate compound onexpression of a reporter gene under control of the expression controlsequences for these enzymes. Assays for the ability of compounds toinhibit control sequences which control the production of, for example,HMG-CoA reductase are set forth in Yagi, Y., et al., Drug DevelopmentResearch (1997) 40:41-47.

[0051] In general, the assays are conducted by contacting a candidatecompound with an expression system wherein said expression systemcomprises a control sequence associated with an enzyme in the isoprenoidsynthesis pathway operably linked to a reporter gene; comparing theexpression of the reporter gene in the presence and absence of thecompound; wherein a decrease in the expression of the reporter gene inthe presence as compared to the absence of the compound indicates thecompound will be useful in treating bone disorders in vertebrates.

[0052] In summary, the statins, known to be inhibitors of HMG-CoAreductase, presumably the rate-limiting step in the isoprenoid pathway,have been shown herein to directly stimulate the formation of bone. Thebisphosphonates, which were shown by Luckman, S. P., et al., J Bone MinRes (1998) (supra) to inhibit prenylation of proteins, and otheraminobisphosphonates which have been shown to inhibit squalene synthase(Aminn, et al., J Lipid Res (1992) 33:1657) have been shown herein tostimulate bone formation directly. Inhibition of the prenylation of rasprotein has also been shown to be related to bone stimulation. Thus,generally, inhibitors of the isoprenoid pathway diagrammed in FIG. 2including, especially, the statins and bisphosphonates, are useful indirect stimulation of bone formation.

[0053] Confirmatory Assays

[0054] The osteogenic activity of the compounds used in the methods ofthe invention which are identified by their effects on isoprenoidsystems, especially HMG-CoA reductase, can be verified using in vitroscreening techniques, such as the assessment of transcription of areporter gene coupled to a bone morphogenetic protein-associatedpromoter or in alternative assays.

[0055] High Throughput Assay

[0056] A rapid throughput screening test for compounds that stimulatebone formation by demonstration that they are capable of stimulatingexpression of a reporter gene linked to a BMP promoter (a surrogate forthe production of bone morphogenetic factors that are endogenouslyproduced) is described in U.S. application Ser. No. 08/458,434, filedJun. 2, 1995, and now allowed, the entire contents of which areincorporated herein by reference. This assay is also described as aportion of a study of immortalized murine osteoblasts (derived from amouse expressing a transgene composed of a BMP-2 promoter drivingexpression of T-antigen) in Ghosh-Choudhery, N., et al., Endocrinology(1996) 137:331-39. In this study, the immortalized cells were stablytransfected with a plasmid containing a luciferase reporter gene drivenby a mouse BMP-2 promoter (-2736/114 bp), and responded in adose-dependent manner to recombinant human BMP-2.

[0057] Briefly, the assay utilizes cells transformed permanently ortransiently with constructs in which the promoter of a bonemorphogenetic protein, specifically BMP-2 or BMP-4, is coupled to areporter gene, typically luciferase. These transformed cells are thenevaluated for the production of the reporter gene product; compoundsthat activate the BMP promoter will drive production of the reporterprotein, which can be readily assayed. Many thousands of compounds havebeen subjected to this rapid screening technique, and only a very smallpercentage are able to elicit a level of expression of reporter gene5-fold greater than that produced by vehicle. Compounds that activatethe BMP promoter fall into groups, where members of each group sharecertain structural characteristics not present in inactive compounds.The active compounds (“BMP promoter-active compounds” or “activecompounds”) are useful in promoting bone or cartilage growth, and thusin the treatment of vertebrates in need of bone or cartilage growth.

[0058] BMP promoter-active compounds can be examined in a variety ofother assays that test specificity and toxicity. For instance, non-BMPpromoters or response elements can be linked to a reporter gene andinserted into an appropriate host cell. Cytotoxicity can be determinedby visual or microscopic examination of BMP promoter- and/or non-BMPpromoter-reporter gene-containing cells, for instance. Alternatively,nucleic acid and/or protein synthesis by the cells can be monitored. Forin vivo assays, tissues may be removed and examined visually ormicroscopically, and optionally examined in conjunction with dyes orstains that facilitate histologic examination. In assessing in vivoassay results, it may also be useful to examine biodistribution of thetest compound, using conventional medicinal chemistry/animal modeltechniques.

[0059] Neonatal Mouse Calvaria Assay (In vitro)

[0060] An assay for bone resorption or bone formation is similar to thatdescribed by Gowen M. & Mundy G., J Immunol (1986) 136:2478-82. Briefly,four days after birth, the front and parietal bones of ICR Swiss whitemouse pups are removed by microdissection and split along the sagittalsuture. In an assay for resorption, the bones are incubated in BGJbmedium (Irvine Scientific, Santa Ana, Calif.) plus 0.02% (or lowerconcentration) β-methylcyclodextrin, wherein the medium also containstest or control substances. The medium used when the assay is conductedto assess bone formation is Fitton and Jackson Modified BGJ Medium(Sigma) supplemented with 6 μg/ml insulin, 6 μg/ml transferrin, 6 ng/mlselenous acid, calcium and phosphate concentrations of 1.25 and 3.0 mM,respectively, and ascorbic acid to a concentration of 100 μg/ml is addedevery two days. The incubation is conducted at 37° C. in a humidifiedatmosphere of 5% CO₂ and 95% air for 96 hours.

[0061] Following this, the bones are removed from the incubation mediaand fixed in 10% buffered formalin for 24-48 hours, decalcified in 14%EDTA for 1 week, processed through graded alcohols; and embedded inparaffin wax. Three μm sections of the calvaria are prepared.Representative sections are selected for histomorphometric assessment ofbone formation or bone resorption. Bone changes are measured on sectionscut 200 μm apart. Osteoblasts and osteoclasts are identified by theirdistinctive morphology.

[0062] Other auxiliary assays can be used as controls to determinenon-BMP promoter-mediated effects of test compounds. For example,mitogenic activity can be measured using screening assays featuring aserum-response element (SRE) as a promoter and a luciferase reportergene. More specifically, these screening assays can detect signalingthrough SRE-mediated pathways, such as the protein kinase C pathway. Forinstance, an osteoblast activator SRE-luciferase screen and an insulinmimetic SRE-luciferase screen are useful for this purpose. Similarly,test compound stimulation of cAMP response element (CRE)-mediatedpathways can also be assayed. For instance, cells transfected withreceptors for PTH and calcitonin (two bone-active agents) can be used inCRE-luciferase screens to detect elevated cAMP levels. Thus, the BMPpromoter specificity of a test compound can be examined through use ofthese types of auxiliary assays.

[0063] In vivo Assay of Effects of Compounds on Murine Calvarial BoneGrowth

[0064] Male ICR Swiss white mice, aged 4-6 weeks and weighing 13-26 gm,are employed, using 4-5 mice per group. The calvarial bone growth assayis performed as described in PCT application WO 95/24211, incorporatedby reference. Briefly, the test compound or appropriate control vehicleis injected into the subcutaneous tissue over the right calvaria ofnormal mice. Typically, the control vehicle is the vehicle in which thecompound was solubilized, and is PBS containing 5% DMSO or is PBScontaining Tween (2 μl/10 ml). The animals are sacrificed on day 14 andbone growth measured by histomorphometry. Bone samples for quantitationare cleaned from adjacent tissues and fixed in 10% buffered formalin for24-48 hours, decalcified in 14% EDTA for 1-3 weeks, processed throughgraded alcohols; and embedded in paraffin wax. Three to five μm sectionsof the calvaria are prepared, and representative sections are selectedfor histomorphometric assessment of the effects on bone formation andbone resorption. Sections are measured by using a camera lucidaattachment to trace directly the microscopic image onto a digitizingplate. Bone changes are measured on sections cut 200 μm apart, over 4adjacent 1×1 mm fields on both the injected and noninjected sides of thecalvaria. New bone is identified by its characteristic woven structure,and osteoclasts and osteoblasts are identified by their distinctivemorphology. Histomorphometry software (OsteoMeasure, Osteometrix, Inc.,Atlanta) is used to process digitizer input to determine cell counts andmeasure areas or perimeters.

[0065] Additional In Vivo Assays

[0066] Lead compounds can be further tested in intact animals using anin vivo, dosing assay. Prototypical dosing may be accomplished bysubcutaneous, intraperitoneal or oral administration, and may beperformed by injection, sustained release or other delivery techniques.The time period for administration of test compound may vary (forinstance, 28 days as well as 35 days may be appropriate). An exemplary,in vivo oral or subcutaneous dosing assay may be conducted as follows:

[0067] In a typical study, 70 three-month-old female Sprague-Dawley ratsare weight-matched and divided into seven groups, with ten animals ineach group. This includes a baseline control group of animals sacrificedat the initiation of the study; a control group administered vehicleonly; a PBS-treated control group; and a positive control groupadministered a compound (non-protein or protein) known to promote bonegrowth. Three dosage levels of the compound to be tested areadministered to the remaining three groups.

[0068] Briefly, test compound, positive control compound, PBS, orvehicle alone is administered subcutaneously once per day for 35 days.All animals are injected with calcein nine days and two days beforesacrifice (two injections of calcein administered each designated day).Weekly body weights are determined. At the end of the 35-day cycle, theanimals are weighed and bled by orbital or cardiac puncture. Serumcalcium, phosphate, osteocalcin, and CBCs are determined. Both leg bones(femur and tibia) and lumbar vertebrae are removed, cleaned of adheringsoft tissue, and stored in 70% ethanol for evaluation, as performed byperipheral quantitative computed tomography (pQCT; Ferretti, J., Bone(1995) 17:353S-64S), dual energy X-ray absorptiometry (DEXA;Laval-Jeantet A., et al., Calcif Tissue Intl (1995) 56:14-18; J. Casez,et al., Bone and Mineral (1994) 26:61-68) and/or histomorphometry. Theeffect of test compounds on bone remodeling can thus be evaluated.

[0069] Lead compounds can also be tested in acute ovariectomized animals(prevention model) using an in vivo dosing assay. Such assays may alsoinclude an estrogen-treated group as a control. An exemplarysubcutaneous dosing assay is performed as follows:

[0070] In a typical study, 80 three-month-old female Sprague-Dawley ratsare weight-matched and divided into eight groups, with ten animals ineach group. This includes a baseline control group of animals sacrificedat the initiation of the study; three control groups (shamovariectomized (sham OVX)+vehicle only; ovariectomized (OVX)+vehicleonly; PBS-treated OVX); and a control OVX group that is administered acompound known to promote bone growth. Three dosage levels of thecompound to be tested are administered to the remaining three groups ofOVX animals.

[0071] Since ovariectomy (OVX) induces hyperphagia, all OVX animals arepair-fed with sham OVX animals throughout the 35 day study. Briefly,test compound, positive control compound, PBS, or vehicle alone isadministered orally or subcutaneously once per day for 35 days.Alternatively, test compound can be formulated in implantable pelletsthat are implanted for 35 days, or may be administered orally, such asby gastric gavage. All animals, including sham OVX/vehicle andOVX/vehicle groups, are injected intraperitoneally with calcein ninedays and two days before sacrifice (two injections of calceinadministered each designated day, to ensure proper labeling of newlyformed bone). Weekly body weights are determined. At the end of the35-day cycle, the animals' blood and tissues are processed as describedabove.

[0072] Lead compounds may also be tested in chronic OVX animals(treatment model). An exemplary protocol for treatment of establishedbone loss in ovariectomized animals that can be used to assess efficacyof anabolic agents may be performed as follows. Briefly, 80 to 100 sixmonth old female, Sprague-Dawley rats are subjected to sham surgery(sham OVX) or ovariectomy (OVX) at time 0, and 10 rats are sacrificed toserve as baseline controls. Body weights are recorded weekly during theexperiment. After approximately 6 weeks (42 days) or more of bonedepletion, 10 sham OVX and 10 OVX rats are randomly selected forsacrifice as depletion period controls. Of the remaining animals, 10sham OVX and 10 OVX rats are used as placebo-treated controls. Theremaining OVX animals are treated with 3 to 5 doses of test drug for aperiod of 5 weeks (35 days). As a positive control, a group of OVX ratscan be treated with an agent such as PTH, a known anabolic agent in thismodel (Kimmel, et al., Endocrinology (1993) 132:1577-84). To determineeffects on bone formation, the following procedure can be followed. Thefemurs, tibiae and lumbar vertebrae 1 to 4 are excised and collected.The proximal left and right tibiae are used for pQCT measurements,cancellous bone mineral density (BMD) (gravimetric determination), andhistology, while the midshaft of each tibiae is subjected to corticalBMD or histology. The femurs are prepared for pQCT scanning of themidshaft prior to biomechanical testing. With respect to lumbarvertebrae (LV), LV2 are processed for BMD (pQCT may also be performed);LV3 are prepared for undecalcified bone histology; and LV4 are processedfor mechanical testing.

[0073] Statin Compounds Useful in the Invention

[0074] The statin compounds useful in the methods and compositions ofthe invention are of the formula:

[0075] wherein X in each of formulas (1) and (2) represents asubstituted or unsubstituted alkylene, alkenylene, or alkynylene linkerof 2-6C;

[0076] Y represents one or more carbocyclic or heterocyclic rings; whenY comprises two or more rings, they may optionally be fused; and

[0077] R′ represents a cation, H or a substituted or unsubstituted alkylgroup of 1-6C. It is understood that if R′ represents a cation withmultiple positive charges, the appropriate number of anions is coupledwith it. Formulas (1) and (2) are, respectively, the unhydrolyzed andhydrolyzed forms of the statin compounds. Preferred substituents on X(or on R′ when R′ is alkyl) are hydroxy, alkoxy, phenyl, amino andalkyl- or dialkylamino.

[0078] The compounds useful in the invention contain at least one andgenerally several chiral centers. Compounds useful in the inventioninclude mixtures of the various stereoisomers and the stereoisomericforms of the compounds individually. Preferred stereoisomers withrespect to the compound of formula (1) are of the formula:

[0079] and the corresponding stereochemistry in the open chain(nonlactone or hydrolyzed) form of formula (2).

[0080] In one set of preferred embodiments, X is unsubstituted; mostpreferably X is selected from the group consisting of —CH₂CH₂—; —CH═CH—;and —C≡C—, especially —CH₂CH₂— and —CH═CH—.

[0081] Preferred embodiments of Y comprise ring systems such asnaphthyl, polyhydro-naphthyl, monohydro- or dihydrophenyl, quinolyl,pyridyl, quinazolyl, pteridyl, pyrolyl, oxazoyl and the like and thereduced or partially reduced forms thereof.

[0082] Preferred embodiments of the substituent Y include those of theformula:

[0083] wherein the ring system may contain π-bonds;

[0084] wherein R¹ is substituted or unsubstituted alkyl;

[0085] each R² is independently a noninterfering substituent;

[0086] R³ is H, hydroxy, or alkoxy (1-6C);

[0087] each m is independently an integer of 0-6, wherein each R² mayreside in any of positions 2-7; and

[0088] p is 0 or 1, depending on the position of any π-bonds.

[0089] Particularly preferred embodiments include those of formulas(4a)-(4f) wherein the upper limit of n is adjusted according to thevalence requirements appropriate for the particular ring system.

[0090] While R¹ may be substituted alkyl, wherein the substituents mayinclude hydroxy, alkoxy, alkylthiol, phenyl, phenylalkyl, and halo,unsubstituted alkyl is preferred. Particularly preferred embodiments ofR¹ are alkyl of 1-6C, including propyl, sec-butyl, t-butyl, n-butyl,isobutyl, pentyl, isopentyl, 1-methylbutyl, and 2-methylbutyl.Particularly preferred are propyl and sec-butyl.

[0091] Preferred embodiments for R² include H, hydroxy, ═O, andsubstituted or unsubstituted lower alkyl (1-4C), in particular methyl,and hydroxymethyl. In the preferred embodiments, each n is independently1 or 2 and preferred positions for substitution are positions 2 and 6(see formula (4)). Particularly preferred embodiments of R² are OH, H,and lower alkyl, in particular CH₃.

[0092] Particularly preferred are embodiments wherein Y is 4(a) or 4(b),and especially embodiments having the substitution pattern indicated informulas 4(g) and 4(h) below.

[0093] As indicated above, the compounds of the invention may besupplied as individual stereoisomers or as mixtures of stereoisomers.Preferred stereoisomers are those of the formulas (4g) and (4h) astypical and appropriate for those represented by the formulas (4a)-(4f).

[0094] Particularly preferred are compounds with the stereochemistry offormulas (4g) and (4h) wherein the noted substituents are the solesubstituents on the polyhydronaphthyl system optionally includingadditional substituents at position 5. Preferred embodiments includethose wherein each of R² is independently OH, CH₂OH, methyl, or ═O.Preferred embodiments of R¹ in these preferred forms are propyl,sec-butyl, and 2-methyl-but-2-yl.

[0095] Additional preferred embodiments of Y are:

[0096] wherein Z is N and both n are 1, and each K comprises asubstituted or unsubstituted aromatic or nonaromatic carbocyclic orheterocyclic ring system which may optionally be spaced from the linkageposition shown in formula (7) by a linker of 1-2C, including —CHOH—,—CO—, and —CHNH₂—, for example. Aromatic ring systems are preferred.Particularly preferred are compounds of formula (7), either as shown orwherein Z is contained in a 6-membered, rather than a 5-memberedaromatic ring. Thus, another group of preferred compounds of theinvention is of formula (7) where Z is N and an additional substituent═CR⁶— is inserted between Z and the bond directed to X, wherein R⁶ islinear, branched or cyclic alkyl. In a preferred embodiment, R⁶ is acyclic alkyl substituent.

[0097] R⁵ is H or linear, branched, cyclic substituted or unsubstitutedalkyl, wherein substituents are preferably hydroxy, alkoxy, phenyl,amino and alkyl- or dialkylamino. Preferably, when R⁵ is alkyl, it isunsubstituted.

[0098] The substituents on the aromatic ring systems or nonaromatic ringsystems of the invention including those designated by K can be anynoninterfering substituents. Generally, the non-interfering substituentscan be of wide variety. Among substituents that do not interfere withthe beneficial effect of the compounds of the invention on boneformation in treated subjects include alkyl (1-6C, preferably loweralkyl 1-4C), including straight, branched or cyclic forms thereof,alkenyl (2-6C, preferably 2-4C), alkynyl (2-6C, preferably 2-4C), all ofwhich can be straight or branched chains and may contain furthersubstituents; halogens, including F, Cl, Br and I; silyloxy, OR, SR,NR₂, OOCR, COOR, NCOR, NCOOR, and benzoyl, CF₃, OCF₃, SCF₃, N(CF₃)₂, CN,SO, SO₂R and SO₃R wherein R is alkyl (1-6C) or is H. Where twosubstituents are in adjacent positions in the aromatic or nonaromaticsystem, they may form a ring. Further, rings not fused to the aromaticor nonaromatic system K may be included as substituents. These rings maybe aromatic and may be substituted or unsubstituted.

[0099] Preferred non-interfering substituents include hydrocarbyl groupsof 1-6C, including saturated and unsaturated, linear or branchedhydrocarbyl as well as hydrocarbyl groups containing ring systems; halogroups, alkoxy, hydroxy, CN, CF₃, and COOR, amino, monoalkyl- anddialkylamino where the alkyl groups are 1-6C. Particularly preferred aresubstituted or unsubstituted aromatic rings.

[0100] Although the number of substituents on a ring symbolized by K maytypically be 0-4 or 0-5 depending on the available positions, preferredembodiments include those wherein the number on a single ring is 0, 1 or2, preferably 0 or 1. However, an exception is that of formula (8),where it is preferred that the aromatic carbocyclic or heterocyclic ringsystem be multiply substituted. In particular, it is preferred that thesubstituents on K in formula (8) themselves contain aromatic rings.Particularly preferred are substituents that contain phenyl rings.

[0101] Particularly preferred are the embodiments of formula (7) or thering expanded form thereof wherein K represents optionally substitutedphenyl. Particularly preferred are compounds wherein R⁵ is isopropyl andK is para fluorophenyl forms.

[0102] The compounds useful in the methods and compositions of theinvention can be synthesized by art-known methods as they resemble aclass of compounds known in the art to behave asantihypercholesterolemic agents. Typical among these is lovastatin,marketed by Merck as Mevacor®. The synthesis of lovastatin and variousanalogs thereof is set forth in U.S. Pat. No. 4,963,538, incorporatedherein by reference. In addition, methods for synthesis of lovastatinand analogous compounds such as compactin (mevastatin), simvastatin, andpravastatin are set forth in U.S. Pat. Nos. 5,059,696; 4,873,345;4,866,186; 4,894,466; 4,894,465; 4,855,456; and 5,393,893, allincorporated herein by reference. Certain of these compounds are alsoproduced by microorganisms as described in U.S. Pat. Nos. 5,362,638;5,409,820; 4,965,200; and 5,409,820, all also incorporated herein byreference. Compounds described as end-products in these documents areuseful in the methods of the invention.

[0103] Additional analogs, including those containing aromaticembodiments of Y, are described in U.S. Pat. No. 5,316,765 incorporatedherein by reference. For example, the preparation of fluvastatin isdescribed in PCT Application WO84/02131. Other compounds are describedin, for example, Roth, B. D., et al., J Med Chem (1991) 34:357-366;Krause, R., et al, J Drug Dev (1990) 3(Suppl. 1):255-257; Karanewsky, D.S., et al., J Med Chem (1990) 33:2952-2956.

[0104] Particularly preferred are hydrolyzed or unhydrolyzed forms oflovastatin (59-0326), mevastatin (59-0327), simvastatin (59-0328)fluvastatin (59-0342), pravastatin, cerivastatin, NK-104 andatorvastatin. Typical forms of these statins are shown in FIGS. 1A and1B.

[0105] The compositions of the invention may also include thebisphosphonates and their analogs. Typically, and preferably, thebisphosphonates are of the formula

[0106] and the pharmaceutically acceptable salts, esters and amidesthereof. Typical salts are those of the inorganic ions, such as sodiumion, potassium ion, calcium ion, magnesium ion and the like; anypharmaceutically acceptable cation may be used. Typical esters are theethyl, methyl, isobutyl, ethylene glycol, and other typicalpharmaceutically acceptable esters; typical amides are the unsubstituted—NH₂ amides as well as the alkyl and dialkyl amides.

[0107] Embodiments of R¹⁰ include halo, OR, SR, NR₂, where R is H oralkyl (1-6C) or alkyl or arylalkyl with optional substitutions.Particularly preferred are the amino-substituted alkyl embodiments.Typically, both R¹⁰ are not identical, although in some embodiments,such as clodronate, both R¹⁰ are halo. Particularly preferred compoundsamong the bisphosphonates are risedronate, alandronate, pamidronate,clodronate and in particular ibandronate. These compounds areparticularly useful in combination with the statins.

[0108] In addition to the statins or other isoprenoid inhibitingcompounds of the invention, the compositions may also include otheragents, including those which inhibit bond resorption such as estrogensor their analogs and compounds of the formula Ar-L-Ar wherein Arrepresents an aryl substituent and L represents a linker.

[0109] The following examples are intended to illustrate but not tolimit the invention.

EXAMPLE 1 High Throughput Screening

[0110] Thousands of compounds have been tested in the assay system setforth in U.S. Ser. No. 08/458,434, filed Jun. 2, 1995, and incorporatedherein by reference. Representative compounds of the invention gavepositive responses, while the majority of (unrelated) compounds areinactive. In this screen, the standard positive control was the compound59-0008 (also denoted “OS8”), which is of the formula:

[0111] In more detail, the 2T3-BMP-2-LUC cells, a stably transformedosteoblast cell line described in Ghosh-Choudhury, et al., Endocrinology(1996) 137:331-39, referenced above, was employed. The cells werecultured using α-MEM, 10% FCS with 1% penicillin/streptomycin and 1%glutamine (“plating medium”), and were split 1:5 once per week. For theassay, the cells were resuspended in a plating medium containing 4% FCS,plated in microtiter plates at a concentration of 5×10³ cells (in 50μl)/well, and incubated for 24 hours at 37° C. in 5% CO₂. To initiatethe assay, 50 μl of the test compound or the control in DMSO was addedat 2×concentration to each well, so that the final volume was 100 μl.The final serum concentration was 2% FCS, and the final DMSOconcentration was 1%. Compound 59-0008 (10 μM) was used as a positivecontrol.

[0112] The treated cells were incubated for 24 hours at 37° C. and 5%CO₂. The medium was then removed, and the cells were rinsed three timeswith PBS. After removal of excess PBS, 25 μl of 1×cell culture lysingreagent (Promega #E153A) was added to each well and incubated for atleast ten minutes. Optionally, the plates/samples could be frozen atthis point. To each well was added 50 μl of luciferase substrate(Promega #E152A; 10 ml Promega luciferase assay buffer per 7 mg Promegaluciferase assay substrate). Luminescence was measured on an automated96-well luminometer, and was expressed as either picograms of luciferaseactivity per well or as picograms of luciferase activity per microgramof protein.

[0113] In this assay, compound 59-0008(3-phenylazo-1H-4,1,2-benzothiadiazine) exhibits a pattern of reactivitywhich is maximal at a concentration of approximately 3-10 μM.Accordingly, other tested compounds can be evaluated at variousconcentrations, and the results compared to the results obtained for59-0008 at 10 μM (which value would be normalized to 100).Alternatively, the reactivity of a compound to be tested can be compareddirectly to a negative control containing no compound.

[0114] A variety of statin compounds (simvastatin designated OS114 or59-0328, hydrolyzed simvastatin, mevastatin designated 59-0327,lovastatin designated 59-0326, fluvastatin designated 59-0342, andpravastatin designated 59-0329) were tested in the in vitro BMP-promoterbased (designated “ABA”) assay (as described at the beginning of thisexample above), and in some experiments were tested also in a controlosteoblast/serum response element (OBSRE) cell-based assay and/or acontrol glucagon assay. In the negative control OBSRE assay, a murineosteoblast cell line, such as CCC-4 expressing a serum response element(SRE)-luciferase reporter gene is used (see WO96/07733). In the negativecontrol glucagon assay, a glucagon receptor-positive BHK cell expressinga CRE-luciferase reporter gene is used (see U.S. Pat. No. 5,698,672).

[0115] The results of three separate determinations in the ABA assay areshown below. Most of the statins (i.e., simvastatin, hydrolyzedsinvastatin, mevastatin, lovastatin, and fluvastatin) exhibited adose-dependent stimulatory effect in the ABA assay, but not in controlassays. In one experiment, simvastatin demonstrated over a 20-foldinduction in the ABA assay, as compared to the non-statin, ABAstimulatory compound 59-0008. ABA Assay Results Sample ConcentrationFold increase % 59-008 over negative μM μg/ml Response controlExperiment 1 Lovastatin 497 200 −22.650 −0.003 59-0326 248.5 100 −22.620−0.001 124.25 50 −22.510 0.003 62.125 25 144.730 7.408 31.0625 12.5153.790 7.809 15.5312 6.25 126.230 6.589 7.76562 3.125 111.730 5.9473.88281 1.5625 67.080 3.970 1.94140 0.78125 35.220 2.559 0.970700.390625 27.830 2.232 0.48535 0.195312 22.840 2.011 Mavastatin 512 200−1.600 0.929 59-0327 256 100 12.460 1.552 128 50 153.110 7.779 64 25152.690 7.760 32 12.5 164.730 8.293 16 6.25 140.260 7.210 8 3.125 99.6705.413 4 1.5625 47.630 3.103 2 0.78125 34.520 2.528 1 0.390625 15.6501.693 0.5 0.195312 25.840 2.144 Simvastatin 477 200 −22.960 −0.00959-0328 238.5 100 −23.010 −0.011 119.25 50 −22.950 −0.008 59.625 25148.310 7.515 29.8125 12.5 131.300 6.768 14.9062 6.25 141.970 7.2377.45312 3.125 135.610 6.957 3.72656 1.5625 99.170 5.356 1.86328 0.7812569.600 4.057 0.93164 0.390625 38.210 2.679 0.46582 0.195312 29.030 2.275Pravastatin 471 200 11.670 1.513 59-0329 235.5 100 7.620 1.335 117.75 505.000 1.220 58.875 25 3.520 1.155 29.4375 12.5 1.110 1.049 14.7187 6.250.920 1.040 7.35937 3.125 0.380 1.017 3.67968 1.5625 −1.120 0.9510.83984 0.78125 −1.980 0.913 0.91992 0.390625 −1.290 0.943 0.459960.195312 −4.390 0.807 Fluvastatin 470 200 −22.700 −0.005 59-0342 235 100−22.570 0.001 117.5 50 −22.660 −0.003 58.75 25 137.500 7.088 29.375 12.5164.450 8.281 14.6875 6.25 165.630 8.333 7.34375 3.125 173.280 8.6723.67187 1.5625 117.980 6.223 1.83593 0.78125 75.160 4.328 0.917960.390625 28.080 2.243 0.45898 0.195312 36.820 2.630 Hydrolyzed 477 200−15.140 −0.0 simvastatin 238.5 100 −15.230 −0.0 119.25 50 44.520 3.959.625 25 200.230 14.3 29.8125 12.5 156.230 11.3 14.90625 6.25 150.26010.9 7.453125 3.125 75.860 6.0 3.726562 1.5625 36.190 3.4 1.8632810.78125 10.430 1.6 0.931640 0.390625 1.520 1.1 0.465820 0.195312 −0.6800.9 Experiment 2 Mevastatin 100 39 88.860 6.9 59-0327 50 19.5 97.420 7.425 9.75 71.630 5.7 12.5 4.875 66.010 5.3 6.25 2.4375 36.210 3.4 3.1251.21875 10.970 1.7 1.5625 0.609375 9.780 1.6 0.78125 0.304687 2.370 1.10.390625 0.152343 0.960 1.0 0.195312 0.076171 −0.090 0.9 0.0976560.038085 2.880 1.1 Simvastatin 477 200 222.180 15.7 59-0328 238.5 100304.100 21.2 119.25 50 189.430 13.5 59.625 25 180.640 13.0 29.8125 12.5152.290 11.1 14.90625 6.25 104.640 7.9 7.453125 3.125 51.660 4.43.726562 1.5625 19.670 2.3 1.863281 0.78125 6.820 1.4 0.931640 0.3906252.600 1.1 0.465820 0.195312 0.310 1.0 Experiment 3 Lovastatin 247 100−32.660 −0.002 59-0326 77.1875 31.25 166.770 6.118 24.12109 9.765625148.030 5.543 7.537841 3.051757 103.260 4.169 2.355575 0.953675 35.3002.083 0.736117 0.298023 10.860 1.333 0.230036 0.093132 3.210 1.0990.071886 0.029103 1.550 1.048 0.022464 0.009094 2.720 1.083 0.0070200.002842 1.050 1.032 0.002193 8.88178E 0.620 1.019 Fluvastatin 235 10060.3 4.14 59-0342 73.4375 31.25 122.53 7.38 22.94921 9.765625 103.7 6.47.171630 3.051757 22.66 2.18 2.241134 0.953674 2.3 1.12 0.7003540.298023 −3.26 0.83 0.218860 0.093132 −2.11 0.89 0.068394 0.029103 −5.180.73 0.021373 0.009094 −3.84 0.8 0.006679 0.002842 −3.26 0.83 0.0020878.88178E −0.19 0.99

[0116] The pravastatin that was tested did not exhibit a dose-dependentstimulatory response, but since this compound was extracted fromformulated pravastatin, it is possible that insufficient and/or inactivecompound was tested.

EXAMPLE 2 In vivo Calvarial Bone Growth Data

[0117] Lovastatin and simvastatin were assayed in vivo according to theprocedure described previously (see “In vivo Assay of Effects ofCompounds on Murine Calvarial Bone Growth,” supra). Simvastatin provideda 1.5 fold increase in the number of osteoblasts.

[0118] In one experiment, vehicle control, bFGF and varying doses ofsimvastatin (59-0328) and lovastatin (designated 59-0326) were tested inthe in vivo calvarial bone growth assay. The results are reported as ameasurement of total bone area (and % increase in area over vehiclecontrol), as shown below. Total Bone Compound Area (μm²) Control 167.7bFGF (12.5 μg/kg/day) 242 (45%) 59-0328 10 mg/kg/day 245 (46%) 59-0328 5mg/kg/day 202 (20%) 59-0328 1 mg/kg/day 172 (2%) 59-0326 10 mg/kg/day239 (42%) 59-0326 5 mg/kg/day 235 (40%) 59-0326 1 mg/kg/day 237 (41%)59-0326 0.1 mg/kg/day 162 (0%)

[0119] Both simvastatin and lovastatin stimulated a dose-dependentincrease in total bone area. At 10 mg/kg/day, the bone stimulatoryeffects of these statins were comparable to the bone growth effectobserved when 12.5 μg/kg/day bFGF was tested in the same assay.

EXAMPLE 3 In vitro Bone Formation

[0120] Selected compounds and appropriate controls were assayed in vitro(ex vivo) for bone formation activity (described above in “NeonatalMouse Calvaria Assay (in vitro)”). Histomorphometrical assessments of exvivo calvaria were carried out using an OsteoMetrics bone morphometrymeasurement program, according to the manufacturer's instructions.Measurements were determined using either a 10- or 20-fold objectivewith a standard point counting eyepiece graticule.

[0121] New bone formation was determined (using a 10X objective) bymeasuring the new bone area formed in one field in 3 representativesections of each bone (4 bones per group). Each measurement was carriedout ½ field distance from the end of the suture. Both total bone and oldbone area were measured. Data were expressed as new bone area in mm².

[0122] Osteoblast numbers were determined by point counting. The numberof osteoblast cells lining the bone surface on both sides of the bonewere counted in one field using a 20×objective. Data were expressed asosteoblast numbers/mm of bone surface.

[0123] Lovastatin and simvastatin and control compounds/factors bFGF andBMP-2, and a vehicle control were tested in the in vitro bone formationassay and the calvaria were analyzed histomorphometrically, as describedabove. Several sets of experimental data are presented below, with %increase over vehicle control values indicated parenthetically.Osteoblast Number: 3 days 7 days Compound Obs/field Obs/field Experiment1 Control 97.25 92.7 59-0328 156 (63%) 154 (68%) BFGF 179.25 (84%) 168.7(82%) BMP-2 127 (31%) 119 (28%) Experiment 2 Control 90.7 59-0328 154(70%) Experiment 3 Control 84 59-326 128 (152%)

[0124] New Bone Area: 3 days 7 days Compound (mm² × 10⁻³) (mm² × 10⁻³)Experiment 1 Control 4.8 7.8 59-0328 8.5 (77%) 17.4 (123%) BFGF 5.7(19%) 18.1 (132%) BMP-2 8.4 (75%) 18.7 (140%) Experiment 2 control 4.059-0328 15.4 (285%) Experiment 3 Control 4.5 590-326 8.2 (182%)

[0125] These data show that lovastatin and simvastatin are as good as,or better than, BMP-2 and bFGF (two “gold standard” agents for bonegrowth; see Wozney J., Molec Reprod Dev (1992) 32:160-67; WO95/24211)for inducing bone formation. As shown in Example 2, in vivo calvarialstudies using lovastatin and simvastatin have provided bone growth dataconsistent with these in vitro observations.

EXAMPLE 4 Effect on Resorption

[0126] The statins and controls were tested in an antiresorptive assay.Briefly, 15 day timed pregnant CD-1 female mice were injected with ⁴⁵Ca(25 μCi/mouse). The calvaria from the 4 day old pups were dissected outand cut in half. The excised half calvaria were placed on metal grids(at the surface) in 1 ml of BGJ medium (Sigma) containing 0.1% BSA withglutamine and Pen/Strep added. The bones were incubated at 37° C. in a5% humidified incubator for a period of 24 h, and then were transferredto wells containing 1 ml medium with factors added (IL-1, PTH, and/ortest compounds). The treated bones were incubated under the aboveconditions for a further 72 h. After this incubation period, the boneswere removed and placed into 20% TCA in a scintillation vial for 1.5 h,and then counted with scintillation fluid. An aliquot of medium (0.4 ml)was also counted. The results were expressed as % ⁴⁵Ca release.

[0127] This assay may be modified by including test compounds/factors orcontrol compounds/factors in the preincubation medium (i.e., during thefirst 24 h). Since most of the osteoclasts are formed in the calvariafollowing the preincubation period, compounds or factors that affectosteoclast formation may have a greater effect during the preincubationperiod.

[0128] In this assay, compound toxicity was indicated by obvious deathof the cells in the periosteal region and within the marrow cavity ofthe bone organ cultures. These cells were characterized by pyknoticnuclei and vacuolated cytoplasm, characteristic of cell necrosis anddistinct from apoptosis.

[0129] Using this assay, simvastatin was tested for its ability toinhibit IL-1 induced bone resorption. Briefly, IL-1 (10⁻¹⁰M) was addedsimultaneously with simvastatin (at 0.1, 1 or 10 μM) during a 72 hincubation period. Bone resorption was determined by measuring ⁴⁵Carelease. IL-1, in the absence of simvastatin, increased ⁴⁵Ca releaseabout 2-fold over control calvaria incubated in the absence of IL-1 orsimvastatin. Simvastatin at 0.1 or 1 μM concentration did not alter IL-1induced ⁴⁵Ca release. However, simvastatin at 10 μM concentrationdecreased IL-1 induced ⁴⁵Ca release.

[0130] Histologic evaluation indicated toxicity at the 10 μMsimvastatin. Previous studies have correlated toxicity with decreased⁴⁵Ca release.

[0131] These data suggest that simvastatin does not inhibit boneresorption at doses effective in the primary screening assay ofExample 1. These results are contrary to reports by R. G. G. Russell andcolleagues that mevastatin inhibits bone resorption in murine calvariaein vitro.

EXAMPLE 5 Systemic Administration of Statins in OVX Models

[0132] Lovastatin and simvastatin were analyzed in vivo using an acuteOVX (prevention model) and/or chronic (treatment model) OVX modelsystem, as described above under “Additional In Vivo Assays”.

[0133] Lovastatin was examined in an acute OVX study. Briefly, 59-0326was orally administered (35, 7, or 1.4 mg/kg/day; once per day for 35days) immediately following ovariectomy. At the end of the study, theanimals' blood and tissues samples were processed, and the followingdata were obtained.

[0134] pQCT analysis of samples removed from animals that received the 7mg/kg/day dose showed a 20% increase in trabecular density and a 14%increase in cortical thickness. Histomorphometric analysis by twoindividuals showed an increase in trabecular bone volume of 110%(p<0.001) or 25% (p=0.0503) in the proximal tibia, and an increase of23% (not significant) in the distal femur. The bone formation rate inthe distal femur was increased 40% (p=0.052) in the 7 mg/kg/day animals.

[0135] Similarly, lovastatin was orally administered (0.1, 1, 5, 10, 20or 40 mg/kg/day; once per day for 35 days) immediately followingovariectomy. Two individuals histomorphometrically analyzed samplesremoved from animals that received the 10 mg/kg/day dose. These analysesindicated an increase in trabecular bone volume of 38% (not significant)or 90% (p<0.02) in the proximal tibia. The bone formation rate in theproximal tibia was determined to have increased 74% (p=0.004) in animalsthat received a 10 mg/kg/day dose or 37% (p<0.008) in animals thatreceived a 1 mg/kg/day dose. In samples from animals that received the 1mg/kg/day dose, mineral apposition rate in the proximal tibia wasincreased 22%.

[0136] Simvastatin was also examined in a chronic OVX study. Briefly,simvastatin was orally administered (0.1, 1, 10, or 50 mg/kg/day; onceper day for 10 weeks) to rats at 6 weeks post-ovariectomy. At the 10mg/kg/day dose, a 114% (not significant) increase in the bone formationrate in the proximal tibia was measured. At the 50 mg/kg/day dose, an86% (not significant) increase in the bone formation rate in theproximal tibia was measured.

EXAMPLE 6 Statin-Mediated Fracture Repair Effects of Test Compounds

[0137] Simvastatin was examined for effects on surgical defects in therabbit radius. Healing of these defects may be assessed by X-ray,histology and biomechanical strength.

[0138] The test compound was weighed out in a microcentrifuge tube, and50 μl of 1.5% sodium alginate solution was added as a carrier. This testsample was vortexed to wet all of the powder. The sample was sonicatedfor 20-30 min, and then vortexed again. Disks were created in the top ofthe microcentrifuge tube (by placing the tube lid or stopper-side down).The indent in the top of the stopper (i.e., lid) was used to form thedisk (7.5 mm diameter). CaCl₂ solution (100 μl of 100 mM) was added tothe sodium alginate/drug solution. The samples were allowed to sit for5-10 min, and then the calcium-alginate disks were carefully removed.The disks were rinsed in a beaker filled with water to rinse off theexcess calcium solution, and were saved in tubes using water as vehicle.All solutions and containers were sterile, and all procedures forpreparations of disks were performed under a laminar flow hood understerile conditions.

[0139] Bone healing was examined as follows. Briefly, six-month old malerabbits were obtained, and were divided into 4 treatment groups (n=3animals/group). The treatment groups received either: 1) placebo; 2)test compound (5 mg/disk); 3) test compound (10 mg/disk); or 4) anautologous bone graft. Animals were anesthetized with rabbit cocktail (1ml/1.5 kg intramuscularly), and the right forelimb was clipped, preppedand draped for aseptic surgery. Anesthesia was maintained usingisofluorane delivered with a face mask. To create a 20 mm gap defect inthe right mid-radius, an incision was made over the lateral aspect ofthe forearm, and an osteotomy was performed with an oscillating bonesaw. The simvastatin or vehicle was applied to the defect and the defectwas closed in layers. No external splinting was needed, as the radius ispaired with the ulna, which functions to allow normal ambulation in therabbit. Disks were cut on strips and inserted in the fracture to coverall the defect. Radiologic evaluation was performed at zero time and at4 weeks.

[0140] Because the vehicle (placebo treatment group) prevented fullhealing in the control group, only X-ray results were obtained andanalyzed. Accordingly, X-ray analysis 4 weeks after initiation oftreatment showed callus formation at the bone treatment site in thetreated (both doses), but not the placebo (vehicle or autologous bonegraft) groups.

EXAMPLE 7 Effect of Ras Inhibition on Bone Formation

[0141] The 2T3 osteoblast progenitor cell line, described byGhosh-Choudhury, et al., Endo (1995) 137:331-339 is BMP-2 responsive andundergoes bone formation spontaneously in culture in vitro. These cellswere stably transfected with the human dominant negative H-ras (Dn-ras)cDNA; this negative mutant gene contains an asparagine substitution atposition 17 and production of this protein effects inhibition ofendogenous ras. The Dn-ras coding sequence was under transcriptionalcontrol of the dexamethasone inducible mouse mammary tumor viruspromoter. The production of the negative ras protein was confirmed byWestern blot.

[0142] The cells were cultured on 24-well plates at a density of2×10⁴/well and cultured with αMEM medium supplemented with 10% SDS fornodule formation assay indicating bone growth. Bone cell differentiationand module formation were monitored using a mineralized module formationassay described by Bharagava, et al., Bone (1986) 8:155-163 using vanKossa stain. The cultures were washed with phosphate buffered formalin,then with water and dehydrated in 70%, 95% and 100% ethanol, 2x each andthen air-dried. The plates were rehydrated before staining; the waterwas removed, a 2% silver nitrate solution was added and the plates wereexposed to sunlight for 20 minutes. The plates were rinsed with waterand 5% sodium thiosulfate was added for 3 minutes and then rinsed.Modified van Gleson stain was used as a counterstain for unmineralizedcollagen matrix. Acid fuschin solution was added for 5 minutes and theplates were then washed and dried for image analysis.

[0143] The area of van Kossa stained nodules was quantified by anautomated image analysis using a video analysis program (JandelScientific, San Raphael, Calif.) linked to a video screen cameraequipped with metallurgical lenses. The nodule structures were alsoanalyzed by transmission electron microscopy.

[0144] When the cells containing the Dn-ras gene were activated byincubation with 10⁻⁷ M dexamethasone, a dramatic and spontaneousformation of mineralized bone nodules resulted. This result did notoccur in wild-type cells in the absence of added BMP. Thus, inhibitionof ras function causes osteoblasts to differentiate and form bone.

EXAMPLE 8 Stimulation of In Vitro Calvarial Bone Formation byBisphosphonates

[0145] Various bisphosphonates were tested for their ability tostimulate calvarial bone formation in vitro in the assay described inExample 3 both alone and in combination with 59-0328 (simvastatin). Theresults were calculated as new bone area in mm²×10⁻³, as above.

[0146] Dose response curves for ibandronate as a typical bisphosphonateshowed a significant increase of bone formation at 10 μM ibandronate.These results are summarized below: New Bone Area (mm² × 10⁻³)Experiment 1 Experiment 2 Experiment 3 Control 3.4 ± 0.5 3.5 ± 0.45 4.8± 0.6 0.01 μM — 3.5 ± 0.43 — 0.1 μM — 3.2 ± 0.56 6.2 ± 0.6 1 μM — 4.9 ±1.2 7.5 ± 0.7 10 μM 8.3 ± 0.7 7.2 ± 0.7 9.5 ± 2.4 100 μM Toxic ToxicToxic 1000 μM Toxic Toxic —

[0147] Other bisphosphonates were tested as shown below; althoughalendronate, pamidronate and clodronate gave negligible effects underthese conditions, risedronate showed significant increases in new bonearea at 10 μM and 100 μM concentrations. New Bone Area (mm² × 10⁻³)Alendronate Pamidronate Clodronate Risedronate Control 3.8 ± 0.5 5.0 ±0.2 3.9 ± 0.5 3.8 ± 0.4 0.1 μM 3.5 ± 0.3 — — — 1 μM 5.2 ± 0.5 — — — 10μM 3.3 ± 0.6 6.0 ± 0.5 4.8 ± 1.3 8.5 ± 0.5 100 μM Toxic 5.8 ± 0.6 4.6 ±0.6 7.3 ± 1.9 1000 μM — 5.2 ± 0.6 3.5 ± 0.5 Toxic

[0148] The effect of ibandronate together with simvastatin was alsotested. The results are shown as follows: New Bone Area (mm² × 10⁻³) IBN(10 μM) IBN (1 μM) Control  4.13 ± 0.035 3.77 ± 0.93 Ibandronate (IBN)6.03 ± 1.56 3.95 ± 0.28 59-0328 (0.2 μM) 6.83 ± 1.54 5.23 ± 0.89 59-0328(0.1 μM) 4.25 ± 0.59  5.1 ± 0.49 IBN + 328 (0.2 μM) 12.1 ± 1.7   7.6 ±0.61 IBN + 328 (0.1 μM) 8.15 ± 0.77 7.82 ± 1.31

[0149] As shown, the combination of 59-0328 with ibandronate wassuperior to the result with either bisphosphonate alone.

[0150] From the foregoing, it will be appreciated that, althoughspecific embodiments of the invention have been described herein forpurposes of illustration, various modifications may be made withoutdeviating from the spirit and scope of the invention. Accordingly, theinvention is not limited except as by the appended claims.

1. A method to enhance bone formation in a vertebrate animal whichmethod comprises administering to a vertebrate subject in need of suchtreatment an amount of a composition comprising a statin compound of theformula:

wherein X in each of formulas (1) and (2) represents a substituted orunsubstituted alkylene, alkenylene, or alkynylene linker of 2-6C; Y isof the formula

or a stereoisomer thereof, wherein R¹ is substituted or unsubstitutedalkyl; each R² is independently H, hydroxy, alkoxy (1-6C) or lower alkyl(1-4C); R³ is H, hydroxy, or alkoxy (1-6C); or Y is of the formula

wherein each n is 1, Z is N, K comprises a substituted or unsubstitutedaromatic carbocyclic or heterocyclic ring system which may optionally bespaced from the linkage position shown in formula (7) by a linker of1-2C, or in formula (7), Z may be spaced from the carbon bonded to X by═CR⁶— wherein R⁶ is H or linear, branded or cyclic alkyl (1-6C), R⁵ is Hor linear, branched or cyclic alkyl, and R′ represents a cation, H or asubstituted or unsubstituted alkyl group of 1-6C, wherein bone formationis effected.
 2. The method of claim 1 wherein X is selected from thegroup consisting of —CH₂CH₂—; —CH═CH—; and —C≡C—.
 3. The method of claim2 wherein Y is of the formula 4(g) or a stereoisomer or mixture ofstereoisomers thereof.
 4. The method of claim 3 wherein R¹ alkyl 4-5C.5. The method of claim 3 wherein each R² is independently H, methyl orhydroxy.
 6. The method of claim 5 wherein each of R² is independently Hor methyl.
 7. The method of claim 2 wherein Y is of formula (7) asshown.
 8. The method of claim 2 wherein Y is of formula (7) where Z isspaced from the carbon bonded to X by ═CR⁶—, wherein R⁶ is H or linear,branched or cyclic alkyl (1-6C).
 9. The method of claim 7 wherein K is asubstituted or unsubstituted carbocyclic aromatic system.
 10. The methodof claim 8 wherein K is a substituted or unsubstituted carbocyclicaromatic system.
 11. The method of claim 9 wherein K is p-fluorophenyl.12. The method of claim 10 wherein K is p-fluorophenyl.
 13. The methodof claim 2 wherein Y is of formula (8).
 14. The method of claim 13wherein K is substituted pyrrole.
 15. The method of claim 14 whereinsaid substitutions comprise aromatic systems.
 16. The method of claim 15wherein said substitutions comprise substituted and unsubstituted phenylgroups.
 17. The method of claim 16 wherein said substitutions comprisep-fluorophenyl and phenyl.
 18. The method of claim 13 wherein K issubstituted pyridyl.
 19. The method of claim 18 wherein the pyridyl is2- pyridyl.
 20. The method of claim 19 wherein the substitutionscomprise alkyl (1-6c) and alkoxy (1-6c).
 21. The method of claim 2wherein said compound is atorvastatin, cerivastatin, lovastatin,mevastatin, simvastatin, fluvastatin, pravastatin or NK-104 inhydrolyzed or unhydrolyzed form.
 22. The method of claim 1 wherein saidsubject is characterized by a condition selected from the groupconsisting of osteoporosis, bone fracture or deficiency, primary orsecondary hyperparathyroidism, periodontal disease or defect, metastaticbone disease, osteolytic bone disease, post-plastic surgery,post-prosthetic joint surgery, and post-dental implantation.
 23. Themethod of claim 1 which further comprises administering to said subjectone or more agents that promote bone growth or that inhibit boneresorption.
 24. A pharmaceutical composition in unit dosage form toenhance bone formation in a vertebrate animal which compositioncomprises a pharmaceutically acceptable excipient and an amount,effective to promote bone formation, of a compound of the formula:

wherein X in each of formulas (1) and (2) represents a substituted orunsubstituted alkylene, alkenylene, or alkynylene linker of 2-6C; Y isof the formula

or a stereoisomer thereof, wherein R¹ is substituted or unsubstitutedalkyl; each R² is independently H, hydroxy, alkoxy (1-6C) or lower alkyl(1-4C); R³ is H, hydroxy, or alkoxy (1-6C); or Y is of the formula

wherein each n is 1, Z is N, K comprises a substituted or unsubstitutedaromatic carbocyclic or heterocyclic ring system which may optionally bespaced from the linkage position shown in formula (7) by a linker of1-2C, or in formula (7), Z may be spaced from the carbon bonded to X by═CR⁶— wherein R⁶ is H or linear, branded or cyclic alkyl (1-6C), R⁵is Hor linear, branched or cyclic alkyl, and R′ represents a cation, H or asubstituted or unsubstituted alkyl group of 1-6C.
 25. A method toidentify a compound that is useful in treating bone disorders invertebrates which method comprises contacting said compound with anassay mixture for the activity of an enzyme in the isoprenoid pathway;determining the activity of the enzyme in the presence as compared tothe absence of said compound; wherein a decrease of activity of saidenzyme in the presence as opposed to the absence of said compoundindicates that the compound will be useful in treating bone disorders invertebrates.
 26. The method of claim 25 wherein the enzyme is aprenylation enzyme.
 27. The method of claim 25 wherein the enzyme isHMG-CoA reductase.
 28. The method of claim 25 wherein the enzymeparticipates in the conversion of mevalonic acid to transgeranylpyrophosphate.
 29. The method of claim 25 wherein the enzyme convertstransgeranyl pyrophosphate to transfamesyl pyrophosphate.
 30. A methodto treat bone disorders in vertebrates which method comprisesadministering to a vertebrate in need of such treatment a compositioncomprising a compound identified by the method of claim 25 .
 31. Amethod to treat bone disorders in vertebrates which method comprisesadministering to a vertebrate in need of such treatment a compositioncomprising a compound identified by the method of claim 27 .
 32. Amethod to identify a compound useful to treat bone disorders invertebrates which method comprises contacting a candidate compound withan expression system wherein said expression system comprises a controlsequence associated with an enzyme in the isoprenoid synthesis pathwayoperably linked to a reporter gene; comparing the expression of thereporter gene in the presence and absence of the compound; wherein adecrease in the expression of the reporter gene in the presence ascompared to the absence of the compound indicates the compound will beuseful in treating bone disorders in vertebrates.
 33. The method ofclaim 32 wherein the enzyme is HMG-CoA reductase.
 34. A method to treatbone disorders in vertebrates which method comprises administering to avertebrate in need of such treatment a composition comprising a compoundidentified by the method of claim 32 .
 35. A method to treat bonedisorders in vertebrates which method comprises administering to avertebrate in need of such treatment a composition comprising a compoundidentified by the method of claim 33 .